| Summary: | Duchenne Muscular Dystrophy (DMD) is a fatal X-linked condition that affects 1 in 4710 boys. Causative mutations in the DMD gene result in a loss of functional expression of the dystrophin protein. Patients usually die in their second or third decade due to cardiac or respiratory failure. Incidence of cardiomyopathy increases with age, such that clinical symptoms are observed in 25% of boys below 6 years of age, but by the teenage years prevalence is 100%. The current treatments are palliative and there are no cures. Numerous cell-based and animal models for DMD exist, but each has its limitations. This includes inter-species differences, incomplete phenocopying of DMD pathophysiology and limited access to diseased human cardiac cells. Therefore, to complement these models, cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) from DMD patients were used to investigate therapeutic strategies and to evaluate the phenotype to better understand the disease. Towards these goals, protocols were optimised for differentiation, characterisation and enrichment of CMs from DMD hiPSC lines. These lines had been derived in our laboratory by four factor lentiviral reprogramming (LIN28, SOX2, NANOG and OCT4) of skin biopsies from boys clinically and genetically diagnosed as having DMD (Dick et al., 2011). Specifically, lines DMD19 and DMD16 carried premature stop codon mutations in exons 35 and 70 respectively, while DMD4 harboured frameshift deletion in exons 48-50 and were used in the study. In the first instance, no CM differentiation was observed. However, manipulation of cell seeding density and BMP, TGFP and WNT signalling pathways enabled efficiencies to be improved to up to 100%. In addition, culture of the hiPSC-CMs in low glucose / high lactate medium lead to preferential survival of the CMs to improve purity to >90%. Improved efficiency of hiPSC-CM production and enrichment provided material to evaluate methods of gene therapy. This included delivery of micro-dystrophin and gene correction of the genomic mutation with RNA guided nucleases (RGNs) in both DMD16 and 19 lines. In each case, restoration of dystrophin protein was observed. In parallel to the gene therapy studies, methods to perform phenotypic analysis were developed. Thus, hiPSC-CMs were analysed for: a) sarcolemma permeability by measuring response of stress on LDH release from cells; b) mitochondrial function in response to different pharmacological challenges by a Seahorse XF assay, and c) Ca2+ profiles, which were quantified using a method termed SALVO (synchronization, amplitude, length and variability of oscillations). Even under severe stress, by treatment with doxorubicin or hypo-osmolarity, no difference was observed in sarcolemma permeability between CMs derived from DMD or healthy hiPSCs. The alterations in mitochondrial function were observed in CMs from DMD hiPSCs relative to healthy CMs. Particularly in DMD16 hiPSCCMs, significant reduction in the ATP production, maximum respiration, spare reserve capacity and non-mitochondrial respiration was observed, while the proton leak was increased. While the data on calcium profile was derived, the number of replicates was not enough to overcome variability in recordings and draw any conclusions. Additional experiments are currently ongoing to confirm whether these phenotypic observations can be rescued by the gene therapy approaches employed. In summary, the work in this thesis has developed optimised protocols for CM differentiation from hiPSCs carrying mutation in the DMD gene, which has been used for pilot investigation into suitability as a supplementary model for investigating phenotype and evaluating genetic therapies. Further development of this in vitro model should lead to increased understanding and progress toward the development of therapies for DMD.
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